Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

Abstract

ABSTRACT␤-N-Acetylhexosaminidases have attracted interest particularly for oligosaccharide synthesis, but their use remains limited by the rarity of enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, genes of 13 ␤-Nacetylhexosaminidases, including 5 from Bacteroides fragilis ATCC 25285, 5 from Clostridium perfringens ATCC 13124, and 3 from Bifidobacterium bifidum JCM 1254, were cloned and heterogeneously expressed in Escherichia coli. The resulting recombinant enzymes were purified and screened for transglycosylation activity. A ␤-N-acetylhexosaminidase named BbhI, which belongs to glycoside hydrolase family 20 and was obtained from B. bifidum JCM 1254, possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through ␤1-3 linkage to the Gal residue of lactose. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation activities of BbhI were studied in detail. With the use of 10 mM pNP-␤-GalNAc or 20 mM pNP-␤-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer (pH 5.8), BbhI synthesized GalNAc␤1-3Gal␤1-4Glc and GlcNAc␤1-3Gal␤1-4Glc at maximal yields of 55.4% at 45°C and 4 h and 44.9% at 55°C and 1.5 h, respectively. The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of ␤1-3 linkage in ␤-N-acetylhexosaminyl lactose synthesis. IMPORTANCEOligosaccharides play a crucial role in many biological events and therefore are promising potential therapeutic agents. However, their use is limited because large-scale production of oligosaccharides is difficult. The chemical synthesis requires multiple protecting group manipulations to control the regio-and stereoselectivity of glycosidic bonds. In comparison, enzymatic synthesis can produce oligosaccharides in one step by using glycosyltransferases and glycosidases. Given the lower price of their glycosyl donor and their broader acceptor specificity, glycosidases are more advantageous than glycosyltransferases for large-scale synthesis. ␤-N-Acetylhexosaminidases have attracted interest particularly for ␤-N-acetylhexosaminyl oligosaccharide synthesis, but their application is affected by having few enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, we describe a microbial ␤-N-acetylhexosaminidase that exhibited strong transglycosylation activity and strict regioselectivity for ␤-N-acetylhexosaminyl lactose synthesis and thus provides a powerful synthetic tool to obtain biologically important GalNAc␤1-3Lac and GlcNAc␤1-3Lac. Oligosaccharides are widely distributed in nature and play a crucial role in many biological events, such as cell structure modulation, cell-cell recognition and communication, and cellmicrobe/toxin interaction and adhesion; therefore, they are promising and important potential therapeutic agents (1-3). However, their use is limited because of the difficulty of large-scale production of oligosaccharides. Their...

Insect-based diet, a promising nutritional source, modulates gut microbiota composition and SCFAs production in laying hens

“…β-N-acetylhexosaminidases (K01207) is the second main class of chitinolytic enzymes that degrade chitooligosaccharides formed by chitinases into monomers 37 . This enzyme is also attracting particular interest for oligosaccharide synthesis, but the sources of this enzyme are rare 38 and the insect-diet fed bacteria might represent an important source of this exogenous chitinase. GlcNAc is deacetylated by N -acetylglucosamine 6-phosphate deacetylase (K01443), an enzymatic function increased in ID group, to glucosamine-6-phosphate and acetate 39 .…”

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

“…In addition, the glycosylation pathway catalyzed by glycosyltransferases is often retarded by feedback inhibition from the nucleoside phosphate that is generated during the reaction . Recently, the biocatalytic synthesis of LNT2 in high yields by a recombinantly produced β‐ N ‐acetylhexosaminidase originating from Bifidobacterium bidifum was reported, but the reaction relied on p NP‐GlcNAc as a donor substrate, which released toxic p ‐nitrophenol ( p NP) as a by‐product of the reaction. In our group, we recently synthesized LNT2 successfully by using N , N ′‐diacetylchitobiose (chitobiose) as a donor and β‐lactose as an acceptor substrate by transglycosylation catalyzed by two novel β‐ N ‐acetylhexosaminidases of metagenomics origin, HEX1 and HEX2 (Scheme ) .…”

Characteristics of an β‐N‐Acetylhexosaminidase from Bacillus sp. CH11, Including its Transglycosylation Activity

“…For the transglycosylation reaction, it is likely that the pH shift is related to the structural changes of the active site as it makes space for the acceptor. Incidentally, the derivatives with a p NP group, such as p NP‐GlcNAc and p NP‐GalNAc, are used as donors by β‐N‐acetylhexosaminidases from many species (Chen et al., ; Ogata et al., ; Rauvolfová et al., ; Singh et al., ). Chitin oligosaccharides such as N, N′‐diacetylchitobiose are available from chitin easily and cheaply by acid hydrolysis.…”

“…Serratia mascescens YS‐1 enzyme also showed high specificity for the dialcohols 1,4‐butanediol, 1,5‐pentanediol and 1,6‐hexanediol, but was less specific toward xylitol. β‐N‐Acetylhexosaminidases from Bifidobacterium bifidum , Aspergillus flavofurcatis , and Aspergillus oryzae were shown to catalyze transglycosylation reactions with lactose, galactose and N‐acetylglucosamine as acceptors in contrast to the action of the CH11 enzyme (Chen et al., ; Rauvolfová et al., ; Singh et al., ).…”

“…Direct inquiries to author Kurakake (E-mail: kurakake@fubac.fukuyama-u.ac.jp). charides, which function as prebiotics, antiadhesive antimicrobials, and immune modulators (Bode, 2012), can be synthesized by transglycosylation. β-N-Acetylhexosaminidase can transfer Nacetylglucosamine from a substrate donor to lactose resulting in a human milk trisaccharide (Chen et al, 2016, Rauvolfová et al, 2004.…”